Methods and compositions relating to Pseudomonas exotoxin proproteins modified for selective toxicity. The exotoxin is modified to be activated by a desired protease by insertion of a protease activatable sequence in the domain II loop. Activation of the proprotein results in formation of the cytotoxic Pseudomonas exotoxin.
Pseudomonas Exotoxin (PE), which binds and enters mammalian cells by receptor-mediated endocytosis, depends on proteolytic cleavage to generate a C-terminal active fragment which translocates to the cell cytosol, ADP-ribosylates elongation factor 2 and inhibits protein synthesis. Mutant versions of PE which cannot be processed appropriately by cells are non-toxic. Furin has been identified as the intracellular protease responsible for this cleavage. Cleavage occurs between arginine 279 and glycine 280 in an arginine-rich loop located in domain II of the toxin. In biochemical experiments, furin-mediated cleavage is evident only under mildly acidic conditions (pH 5.5). Recently, Garten et al., (EMBO J, 14(11):2424-35 (1995)) have proposed that sequences in the cytoplasmic tail of furin are responsible for its cycling to the cell surface and re-entry through the endosomal compartment. Since PE enters cells via the alpha 2-macroglobulin receptor/Low density lipoprotein receptor-related protein (LRP), it is likely that this receptor delivers PE to an acidic endosomal compartment where it is cleaved by furin. PE is broadly cytotoxic because most mammalian cells and tissues express both LRP and furin. In vivo, the injection of native PE produces profound liver toxicity.
PE has been crystallized and its three dimensional structure determined by X-ray diffraction analysis (Allured et al., Proc. Natl. Acad. Sci., 83:1320-1324 (1986)). PE comprises four structural domains: the N-terminal domain (domain Ia) mediates binding to LRP, a second domain (domain II) has the protease processing site and sequences necessary for translocation to the cytosol; a third domain (domain Ib) has no identified function; and a fourth, C-terminal domain (domain III), has ADP-ribosylating activity and an ER retention sequence.
The existing strategy for targeting the cell-killing activity of PE to cancer cells is to delete the DNA encoding the cell binding domain and replace it with cDNAs encoding binding ligands or antibody fragments that recognize cancer-related cell surface determinants. Surface binding then mediates the internalization of PE-immunotoxins to a furin-containing compartment where the appropriate C-terminal fragment is generated. Since most cancer cells express furin, this cleavage-activation step does not contribute to the selectivity of immunotoxin action.
Data from Phase I/III clinical trials indicate that the low level expression of target antigens on normal cells represents a significant impediment to the success of immunotoxin-based therapeutics. This problem may be particularly relevant for the treatment of solid tumors, where individual cancer cells are difficult to access and high levels of immunotoxins must be maintained for prolonged periods.
Cancer cells frequently express high levels of certain proteases including metalloproteinases, serine proteases and various lysosomal enzymes. These function both to promote metastatic spread of cells and to release (from precursors and binding proteins) growth factors locally. Prostate specific antigen (PSA), is a kallikrein-like protease which normally cleaves Semenogelin I and II at several sites but is often elevated to very high levels in patients with prostate cancer. Further, several recent reports suggest that PSA is also expressed in breast cancer tissue. In prostate cancer, PSA is found circulating in serum as a complex with CTI but apparently is active locally where it confers some survival advantage to prostate cancer cells by virtue of its ability to degrade matrix proteins and release insulin-like growth factor from its binding proteins.
Pseudomonas Exotoxin A (xe2x80x9cPExe2x80x9d) is translocated into the cytosol after a furin recognition site in domain II is cleaved by furin. Protease-activatable PE-like proproteins are engineered to replace the furin recognition site by a site recognized by a protease made or secreted by a cell targeted for death, for example, a cancer cell. Upon cleavage by the target protease, the PE-like proprotein is translocated into the cytosol where the toxin""s ADP-ribosylating activity kills the cell by interfering with polypeptide elongation.
The PE-like proproteins of this invention offer several advantages. First, because they are activated by a target protease, and not by furin, their toxicity is significantly more cell-specific than native PE. Second, when the cysteine-cysteine loop of PE domain II is cleaved, the disulfide bond, before it is reduced, holds the cell-recognition domain attached to the rest of PE. Many cancer cells secrete cell-specific proteases that tend to accumulate around the cell. For example, prostate cancer cells secrete prostate specific antigen. Therefore, the proproteins of this invention may be cleaved before entering the target-cell. However, the protease activatable sequence is introduced into the cysteine-cysteine loop of a domain II-like sequence of the proprotein. Therefore, the cell recognition domain is still attached upon cleavage of the proprotein outside the cell, and still is available to bind to a cell surface receptor for subsequent endocytosis. Third, by selecting a proper cell recognition domain, the toxins can be targeted to bind to specific cell types. For example, the modified PE proprotein can be administered as an immunotoxin to further increase its selective toxicity to the desired cells.
The protease is typically expressed within a mammalian cell. The protease within the cell may be native to that cell type, or the cell may be engineered to express a non-native protease. Thus, the present invention has both ex vivo and in vivo utility. Ex vivo utilities include selective elimination of cultured mammalian cells expressing the protease which cleaves the protease activatable sequence. Nucleic acids encoding a PE proprotein can be used as vectors. Disruption of the PE proprotein coding sequence with a nucleic acid insert allows mammalian cells transfected with the vector to survive. Cells transfected with the PE proprotein vector are eliminated. The protease activatable sequence can be modified to be sensitive to the desired protease.
In vivo utilities include increased selective toxicity of PE to particular mammalian cells (e.g., cancer cells) which express proteases which are substantially exclusive to those cells.
In one aspect this invention provides a protease-activatable Pseudomonas exotoxin A-like (xe2x80x9cPE-likexe2x80x9d) proprotein comprising: (1) a cell recognition domain of between 10 and 1500 amino acids that binds to a cell surface-receptor; (2) a modified PE translocation domain comprising an amino acid sequence sufficiently homologous to domain II of PE to effect translocation to a cell cytosol upon proteolytic cleavage, wherein the translocation domain comprises a cysteine-cysteine loop that comprises a protease activatable sequence cleavable by a protease and wherein the cysteine-cysteine loop is substantially un-activatable by furin; (3) optionally, a PE Ib-like domain comprising an amino acid sequence up to 1500 amino acids; (4) a cytotoxicity domain comprising an amino acid sequence substantially homologous to domain III of PE, the cytotoxicity domain having ADP-ribosylating activity; and (5) an endoplasmic reticulum (xe2x80x9cERxe2x80x9d) retention sequence.
In one embodiment of a PE-like proprotein, the modified PE translocation domain has a PE domain II sequence (amino acids 253-364 of SEQ ID NO:1) modified with amino acids substitutions introducing the protease activatable sequence so as to cause cleavage by the protease between amino acids 279 and 280. In another embodiment the protease activatable sequence is cleavable by a protease secreted by a cancer cell. In another embodiment, the cell recognition domain comprises an antibody that specifically binds to a cancer cell surface marker. In another embodiment, the protease activatable sequence is cleavable by prostate specific antigen (xe2x80x9cPSAxe2x80x9d), urokinase, neutral endoprotease, stromelysin, collagenase, cathepsin B, or cathepsin D. In another embodiment, the PE Ib-like domain, the cytotoxicity domain and the ER retention. sequence together have the sequence of domains Ib and III of native PE. In other embodiment, the cell recognition domain is coupled to the modified translocation domain through a peptide bond (i.e., as a fusion protein) or through a chemical linkage.
In another aspect, this invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of a protease-specific Pseudomonas exotoxin A-like (xe2x80x9cPE-likexe2x80x9d) proprotein of this invention.
In another aspect, this invention provides a recombinant polynucleotide comprising a nucleotide sequence encoding a protease-specific Pseudomonas exotoxin A-like (xe2x80x9cPE-likexe2x80x9d) proprotein of this invention. In one embodiment, the recombinant polynucleotide is an expression vector further comprising an expression control sequence operatively linked to the nucleotide sequence.
In another aspect, this invention provides a method for killing a target cell comprising contacting the cell with a protease-specific Pseudomonas exotoxin A-like (xe2x80x9cPE-likexe2x80x9d) proprotein of this invention. In one embodiment, the cancer cell is, without limitation, a prostate cancer cell, a breast cancer cell or a colon cancer cell.
In another aspect, this invention provides a method for therapeutically treating a subject suffering from cancer comprising administering to the subject a protease-specific Pseudomonas exotoxin A-like (xe2x80x9cPE-likexe2x80x9d) proprotein of this invention. More specifically, the PE-like proprotein comprises a protease activatable sequence that is cleavable by an enzyme produced by the cancer cell. The PE-like proprotein can be administered as a pharmaceutical composition.